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I am encountering the problem while generating the constraints form N-15 edit AND C-13 EDIT NOESY files that very few constraints 200 out of 2400 peaks . Calibration is OK . chemical shift file is good agreement with secondary structure of know crystal structure .

Completeness of assignment:

Completeness of all atom assignment is 80.74 %.

Completeness of proton atom assignment is 82.50 %.

Completeness of heavy atom assignment is 74.56 %.

what can be the other possible errors ?

could you please make some helpful comments


Srinivas P

(Merry Christmas & Happy New year to all )

asked Dec 23 '11 at 10:30

sri's gravatar image


updated Dec 27 '11 at 09:53

I observed that i didn't integrate the peaks properly ! ( i think it is better to remove the diagonal peaks while integrate the peaks ! - sri (Jan 08 '12 at 23:10)

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82% assignment of protons is VERY low. Extreme caution would be advisable before proceeding to the restraint generation stage before completion of proton assignments.

For proteins, many observable peaks in the NOESY correspond to intra-residue and sequential-residues and may not be of much use for tertiary structure determination. Since the crystal structure is available, you may check if you are limited by sensitivity by identifying the longest distance in the crystal structure for non-sequential NOe's involving backbone protons observable in your spectra. If this value is significantly lower than 0.45 nm, then you may be able to obtain more restraints by increasing the S/N of your spectra. The S/N ratio for your spectra may be increased by

  • increase of mixing time (Caution !!),
  • increase of protein concentration,
  • increase of field strength (preferable),
  • use of cryoprobe/10 mm probe (instead of the standard 5mm RT),
  • change of temperature (optimum temperature of NOESY is usually not always the same as the optimum temperature for 1D or HSQC spectra),
  • change of pH, etc.

    In addition, proper choice of the pulse sequences and optimization of the delays to match the observed relaxation times is often critical for obtaining good quality X-edited NOESY spectra.

2D-NOESY (homonuclear) spectra often contain many non-sequential NOe peaks that are not observable in other NOESY spectra, most of these are usable as restraints if a low resolution structure is used as a filter to assist in the assignment process.

Some proteins may adopt a specific conformation in the solid (crystal), but may have substantial local flexibility in solution. in such cases, especially for helical proteins, it may be difficult to observe many non-sequential NOe's. Restraints from other types of data, such as J-coupling, are less sensitive to low amplitude local dynamical motion.


answered Jan 15 '12 at 11:05

sekhar%20Talluri's gravatar image

sekhar Talluri

updated Jan 15 '12 at 11:19

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